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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 August 2005 to 04 October 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies , which do not affect the quality of relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Identification: EA-3098
CAS number: 55349-01-4
Description: white powder
Batch: P-32681
Purity: not indicated by the sponsor, treated as 100% pure
Storage: room temperature in the dark
Stability under storage conditions: stable
Expiry date: June 6th 2009

Method

Target gene:
Not applicable
Species / strain
Species / strain:
lymphocytes: human
Details on mammalian cell lines (if applicable):
- Type and identity of media:
Culture medium: Culture medium consisted of RPMI 1640 medium, supplemented with 20% (v/v) heat-inactivated foetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/ml and 50 µg/ml respectively) and 30 U/ml heparin.

Lymphocyte cultures: Whole blood (0.4 ml) treated with heparin was added to 5 ml or 4.8 ml culture medium (in the absence and presence of S9-mix respectively). Per culture 0.1 ml (9 mg/ml) phytohaemagglutinin was added.
Additional strain characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital and ß-naphthoflavone-induced rat liver S9
Test concentrations with justification for top dose:
Dose range-finding test: 0.3, 1.0, 3.0, 10, 33 and 100 µg/ml
First cytogenetic assay: 3, 10 and 33 µg/ml (with and without S9-mix)
Second cytogenetic assay: 10, 33 and 100 µg/ml (without S9-mix)
3, 10 and 33 µg/ml (with S9-mix)
Vehicle:
- Vehicle(s)/solvent(s) used: Ethano.
EA-3098 was suspended in ethanol absolute. The final concentration of the solvent in the culture medium amounted to 1.0% (v/v).
Controlsopen allclose all
Negative controls:
no
Solvent controls:
yes
Remarks:
(solvent treatment groups were used as the vehicle control )
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Without metabolic activation (-S9-mix)
Negative controls:
no
Solvent controls:
yes
Remarks:
(solvent treatment groups were used as the vehicle control )
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
With metabolic activation (+S9 mix)
Details on test system and conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hours
- First cytogenetic assay: With and without S9-mix: 3 h exposure time, 24 h fixation time.
- Second cytogenetic assay: Without S9 mix: 24 h and 48 h exposure time, 24 and 48 h fixation time).
With S9 mix: 3 h exposure time, 48 h fixation time.


SELECTION AGENT (mutation assays): not applicable
SPINDLE INHIBITOR (cytogenetic assays): colchicine (0.5 µg/ml medium).
STAIN (for cytogenetic assays): 5% (v/v/) Giemsa solution in tap water

NUMBER OF REPLICATIONS: Two

NUMBER OF CELLS EVALUATED: The mitotic index of each culture was determined by counting the number of metaphases per 1000 cells . To analyse slides for chromosome aberrations, 100 metaphase chromosome spreads per culture were examined.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy



Evaluation criteria:
The test substance was considered positive (clastogenic) if:
a) It induced a dose-related statistically significant (Chi-square test), P < 0.05) increase in the number of cells with chromosome aberrations.
b) A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absnece of a clear dose-response relationship.

A test substance was considered negative (not clastogenic) in none of the tested concentrations induced a statistically significant (Chi-square test, P < 0.05) increase in the number of cells with chromsome aberrations.
Statistics:
The incidence of aberrant cells (cells with one or more chromosome aberrations, inclusive or exclusive gaps) for each exposure group was compared to that of the solvent control using chi-square statistics.

Results and discussion

Test results
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
(> 33 µg/ml)
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
DOSE RANGE FINDING TEST:
At a concentration of 33 µg/ml EA-3098 precipitated in the culture medium. In the dose range finding study, at the 3 h exposure time, blood cultures were treated with 0.3, 1, 3, 10 and 33 µg EA-3098/ml culture medium with and without S9-mix. At the 24 hand 48 h continuous exposure time blood cultures were treated with 0.3,1,3,10,33 and 100 µg EA-3098/ml culture medium without S9-mix. EA-3098 was tested beyond the limit of solubility to obtain adequate toxicity data.

Table 1 (attached background material) shows the mitotic index of cultures treated with various EA-3098 concentrations or with the negative control substance.

FIRST CYTOGENETIC ASSAY:
Based on the results of the dose range finding test, the following dose levels were selected for the cytogenetic assay:
WIth and without S9-mix: 3, 10 and 33 µg/ml

Table 2 (attached background material) shows the mitotic index of cultures treated with various EA-3098 concentrations or with the positive or negative control substances.

All dose levels were selected for scoring of chromosome aberrations. Both in the absence and presence of S9-mix EA-3098 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations (Tables 3, 4 - attached backgroud material).

Both in the absence and presence of S9-mix EA-3098 did not increase the number of polyploid cells and cells with endoreduplicated chromosomes.

SECOND CYTOGENETIC ASSAY:
To obtain more information about the possible clastogenicity of EA-3098, a second cytogenetic assay was performed in which human lymphocytes were continuously exposed to EA-3098 in the absence of S9 mix for 24 or 48 hours. In the presence of S9-mix, cells were fixed after 48 hours following a 3 hour exposure to EA-3098. The following dose levels were selected for the second cytogenetic assay:

Without S9-mix: 10, 33 and 100 µg/ml
With S9-mix: 3, 10 and 33 µg/ml

Table 5 (attached background material) shows the mitotic index of cultures treated with various EA-3098 concentrations or with the positive or negative control substances.

All dose levels were selected for scoring of chromosome aberrations. Both in the absence and presence of S9-mix EA-3098 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations (Tables 6-8- attached backgroud material).

Both in the absence and presence of S9-mix EA-3098 did not increase the number of polyploid cells and cells with endoreduplicated chromosomes.















Any other information on results incl. tables

Evaluation of the results

The mitotic indices of cultures treated with various EA-3098 concentrations or with the negative control substances are presented in Tables 1, 2 and 5 (see attached background material). The scores for the number of aberrant cells (gaps included and excluded) and the number of the various types of chromosome aberrations at the various concentrations of EA-3098 are presented in Tables 3, 4 and 6-8 (see attached background material). Duplicate cultures are indicated by A and B.

The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. The number of polyploidy cells and cells with endoreduplicated chromosomes found in the solvent control cultures was within the laboratory historical control data range. The positive control chemicals (MMC-C and CP) both produced statistically significant increases in the frequency of aberrant cells. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Both in the absence and presence of S9 -mix EA-3098 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in two independent experiments.

No effects of EA-3098 on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that EA-3098 does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report.

Finally, it is concluded that this test is valid and that EA-3098 is not clastogenic in human lymphocytes under the experimental conditions described in this report.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative (with and without metabolic activation)

The test material is not clastogenic in human lymphocytes under the experimental conditions in the study.
Executive summary:

The ability of EA-3098 to induce chromosome aberrations in cultured peripheral human lymphocytes was evaluated in a study conducted to the following guidelines: OECD Guideline 473 and EU Method B.10. The possible clastogenicity was tested in two independent experiments.

In the first cytogenetic assay, EA-3098 was tested up to 33 µg/ml for a 3 h exposure time with a 24 hour fixation time in the absence and presence of 1.8 % (v/v) S-9 fraction. EA-3098 precipitated in the culture medium at this dose level.

In the second cytogenetic assay, EA-3098 was tested up to 100 µg/ml for a 24 -h and 48 -h continuous exposure time with a 24 -h and 48 -h fixation time in the absence of S9 -mix. In the presence of S9 -mix EA-3098 was tested up to 33 µg/ml for a 3 -h exposure time with a 48 -h fixation time. EA-3098 precipitated in the culture medium at these dose levels.

Positive control chemicals, mytomycin-C and cyclophosphamide, both produced statistically significant increases in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

EA-3098 did not induce a statistically significant or biologically relvenat increase in the number of cells with chromosomeome aberrations in the absence or presence of S9 -mix, in two independently repeated experiments.

No effects of EA-3098 on the number of polyploid cells and cells with andoreplicated chromosomes were observed both in the absence and presence of S9 -mix. Therefore it can be concluded that EA-3098 does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditins described in this report.

It is concluded that this test is valid and that EA-3098 is not clastogenic to human lymphocytes under the experimental conditions in the study.